Details of a Researcher - Fujiwara, Kei

Papers - Fujiwara, Kei

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Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution

Sato G., Kinoshita S., Yamada T.G., Arai S., Kitaguchi T., Funahashi A., Doi N., Fujiwara K.

ACS Synthetic Biology (ACS Synthetic Biology) 13 ( 5 ) 1572 - 1581 2024.05

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

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Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space

Nishikawa S., Sato G., Takada S., Kohyama S., Honda G., Yanagisawa M., Hori Y., Doi N., Yoshinaga N., Fujiwara K.

Advanced Science (Advanced Science) 11 ( 6 ) 2024.02

Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

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Phase-Separated Giant Liposomes for Stable Elevation of α-Hemolysin Concentration in Lipid Membranes

Kobayashi M., Noguchi H., Sato G., Watanabe C., Fujiwara K., Yanagisawa M.

Langmuir (Langmuir) 39 ( 32 ) 11481 - 11489 2023.08

ISSN 07437463

Staphylococcus aureus α-hemolysin (αHL) is one of the most popular proteins in nanopore experiments within lipid membranes. Higher concentrations of αHL within the lipid membrane are desirable to enhance the mass transport capacity through nanopores. However, the reconstitution of αHL at high concentrations is associated with the problem of membrane lytic disruption. In this study, we present a method that effectively increases αHL concentration while maintaining membrane stability. This method is achieved by using phase-separated giant liposomes, where coexisting liquid-disordered (Ld) and liquid-ordered phases (Lo) are enriched in unsaturated lipids and saturated lipids with cholesterol (Chol), respectively. Fluorescence observation of αHL in liposomes revealed that the presence of Chol facilitates αHL insertion into the membrane. Despite the preferential localization of αHL in the Ld phase rather than the Lo phase, the coexistence of both Lo and Ld phases prevents membrane disruption in the presence of concentrated αHL. We have explained this stabilization mechanism considering the lower membrane tension exhibited by phase-separated liposomes compared to homogeneous liposomes. Under hypertonic conditions, we have successfully increased the local concentration of αHL by invagination of the lipid-only region in the Ld phase, leaving αHL behind. This method exhibits potential for the reconstitution of various nanochannels and membrane proteins that prefer the Ld phase over the Lo phase, thus enabling the production of giant liposomes at high concentrations and the replication of the membrane-crowding condition observed in cells.

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Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide

Nakamura M., Fujiwara K., Doi N.

Journal of Nanobiotechnology (Journal of Nanobiotechnology) 20 ( 1 ) 2022.12

Background: Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. Results: We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. Conclusion: These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent.

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Controlling the Periodicity of a Reaction-Diffusion Wave in Artificial Cells by a Two-Way Energy Supplier

Takada S., Yoshinaga N., Doi N., Fujiwara K.

ACS Nano (ACS Nano) 16 ( 10 ) 16853 - 16861 2022.10

ISSN 19360851

Reaction-diffusion (RD) waves, which are dynamic self-organization structures generated by nanosize molecules, are a fundamental mechanism from patterning in nano- and micromaterials to spatiotemporal regulations in living cells, such as cell division and motility. Although the periods of RD waves are the critical element for these functions, the development of a system to control their period is challenging because RD waves result from nonlinear physical dynamics under far-from-equilibrium conditions. Here, we developed an artificial cell system with tunable period of an RD-driven wave (Min protein wave), which determines a cell division site plane in living bacterial cells. The developed system is based on our finding that Min waves are generated by energy consumption of either ATP or dATP, and the period of the wave is different between these two energy suppliers. We showed that the Min-wave period was modulated linearly by the mixing ratio of ATP and dATP and that it was also possible to estimate the mixing ratio of ATP and dATP from the period. Our findings illuminated a previously unidentified principle to control the dissipative dynamics of biomolecules and, simultaneously, built an important framework to construct molecular robots with spatiotemporal units.

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Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology

Yanagisawa M., Watanabe C., Yoshinaga N., Fujiwara K.

Langmuir (Langmuir) 38 ( 39 ) 11811 - 11827 2022.10

ISSN 07437463

Polymer micromaterials in a liquid or gel phase covered with a surfactant membrane are widely used materials in pharmaceuticals, cosmetics, and foods. In particular, cell-sized micromaterials of biopolymer solutions covered with a lipid membrane have been studied as artificial cells to understand cells from a physicochemical perspective. The characteristics and phase transitions of polymers confined to a microscopic space often differ from those in bulk systems. The effect that causes this difference is referred to as the cell-size space effect (CSE), but the specific physicochemical factors remain unclear. This study introduces the analysis of CSE on molecular diffusion, nanostructure transition, and phase separation and presents their main factors, i.e., short- and long-range interactions with the membrane surface and small volume (finite element nature). This serves as a guide for determining the dominant factors of CSE. Furthermore, we also introduce other factors of CSE such as spatial closure and the relationships among space size, the characteristic length of periodicity, the structure size, and many others produced by biomolecular assemblies through the analysis of protein reaction-diffusion systems and biochemical reactions.

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Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells

Sakura Takada, Natsuhiko Yoshinaga, Nobuhide Doi, Kei Fujiwara

Science Advances (Science Advances) 8 ( 23 ) eabm8460 2022.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a substantial role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a previously unidentified role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict an unexplored mechanism in intracellular spatiotemporal pattern formations.

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Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins

Suzuki M., Iwaki K., Kikuchi M., Fujiwara K., Doi N.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 586 63 - 67 2022.01

ISSN 0006291X

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high β-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the β-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a β-strand and that the subsequent parallel β-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

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Activation of a diluted E. coli cell-free transcription-translation system within liposomes by hypertonic concentration

Matsui Y., Akui T., Doi N., Fujiwara K.

STAR Protocols (STAR Protocols) 2 ( 4 ) 2021.12

We present a protocol for activating protein synthesis in liposomes encapsulating a diluted E. coli cell extract-based TX-TL (transcription-translation) system by hypertonic concentration. Protein expression is turned on in the liposome-encapsulated TX-TL system by simple treatment with a concentrated external solution. The expression of sfGFP is demonstrated here, but it can be applied to other proteins. This protocol can be applied to the development of artificial cells utilizing the switch-on mechanism to activate protein expression, responding to the outer environment. For complete details on the use and execution of this protocol, please refer to Akui et al. (2021).

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Transcription-translation of theEscherichia coligenome within artificial cells

Deyama T., Matsui Y., Chadani Y., Sekine Y., Doi N., Fujiwara K.

Chemical Communications (Chemical Communications) 57 ( 80 ) 10367 - 10370 2021.10

ISSN 13597345

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System concentration shift as a regulator of transcription-translation system within liposomes

Akui T., Fujiwara K., Sato G., Takinoue M., Nomura S.i.M., Doi N.

iScience (iScience) 24 ( 8 ) 2021.08

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

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大学の枠を越えたオンライン生体分子デザインコンペティションの取り組み

7. 村田智,葛谷明紀,藤原慶,平順一,川又生吹,佐藤佑介,瀧ノ上正浩,野村M.慎一郎,角五彰,堀豊,安部桂太

工学教育 (日本工学教育協会) 69 ( 4 ) 31 - 39 2021.08

Switching ON of Transcription-Translation System Using GUV Fusion by Co-supplementation of Calcium with Long-Chain Polyethylene Glycol

Uwaguchi Y., Fujiwara K., Doi N.

ChemBioChem (ChemBioChem) 22 ( 13 ) 2319 - 2324 2021.07

ISSN 14394227

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.

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A relationship between ntp and cell extract concentration for cell-free protein expression

Takahashi K., Sato G., Doi N., Fujiwara K.

Life (Life) 11 ( 3 ) 2021.03

The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

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Conformational equilibrium of MinE regulates the allowable concentration ranges of a protein wave for cell division

Kohyama S., Fujiwara K., Yoshinaga N., Doi N.

Nanoscale (Nanoscale) 12 ( 22 ) 11960 - 11970 2020.06

ISSN 20403364

The Min system for determining the cell division position at the center in bacteria has a unique character that uses a protein wave (Min wave) that emerges from its components (MinD and MinE). The Min wave emerges under the coupling of chemical reactions and molecular diffusions of MinDE and appears when the concentrations of MinD and MinE are similar. However, the nanoscale mechanism to determine their concentration ranges has remained elusive. In this study, by using artificial cells as a mimic of cells, we showed that the dominant MinE conformations determined the allowable concentration ranges for the emergence of the Min wave. Furthermore, the deletion of the membrane-binding region of MinE indicated that the region was essential for limiting the concentration ranges to be narrower. These findings illustrate a parameter tuning mechanism underlying complex molecular systems at the nanoscale for spatiotemporal regulation in living cells and show a possibility that the regulation of the equilibrium among molecular conformations can work as a switch for cell division.

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Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Oyobiki R., Fujiwara K., Watanabe T., Einaga Y., Matsumoto Y., Miki N., Doi N.

Lab on a Chip (Lab on a Chip) 20 ( 4 ) 852 - 861 2020.02

ISSN 14730197

We report the development of a micro total analysis system (μTAS) based on electrochemical measurements and dielectrophoretic sorting for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzymes immobilized on microbeads, together with their encoding DNA, can be measured with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. The 30 nL droplets containing microbead-displayed genes of enzymes with higher activity can then be recovered by dielectrophoretic sorting. Previously, we developed the NAD(P)H-measuring device containing the BDD electrode for high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. In this study, we fabricated an encapsulating device and a droplet-sorting device for nanoliter-size droplets, for the first time, and then combined these three devices to construct a μTAS for directed evolution of NAD(P)-dependent oxidoreductases. We confirmed that this system works by proof-of-principle experiments and successfully applied this system for screening of randomized libraries of NAD-dependent dehydrogenases.

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Dielectrophoretic sorting system for screening of NAD(P)-dependent oxidoreductases.

Kanai Y., Shimokihara S., Shitara S., Oyobiki R., Watanabe T., Einaga Y., Wakamatsu K., Matsumoto Y., Fujiwara K., Doi N.

21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 (21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017) 1383 - 1384 2020

Here we report the development of a flow injection analysis system based on electrochemical measurements and dielectrophoresis for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzyme which is immobilized on microbeads together with its encoding DNA can be measured with a boron-doped diamond (BDD) electrode in each compartment (~30 nL) of the microfluidic system, and the large droplets (~30 nL) containing genes of enzymes with higher activity can be recovered by dielectrophoretic sorting.

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells

Kohyama S., Yoshinaga N., Yanagisawa M., Fujiwara K., Doi N.

eLife (eLife) 8 2019.07

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-tovolume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

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Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system

Yoshida A., Kohyama S., Fujiwara K., Nishikawa S., Doi N.

Chemical Science (Chemical Science) 10 ( 48 ) 11064 - 11072 2019

ISSN 20416520

Spatiotemporal patterning is a fundamental mechanism for developmental differentiation and homeostasis in living cells. Because spatiotemporal patterns are based on higher-order collective motions of elements synthesized from genes, their behavior dynamically changes according to the element amounts. Thus, to understand life and use this process for material application, creation of artificial cells with time development of spatiotemporal patterning by changes of element levels is necessary. However, realizing coupling between spatiotemporal patterning and synthesis of elements in artificial cells has been particularly challenging. In this study, we established a system that can synthesize a patterning mechanism of the bacterial cell division plane (the so-called Min system) in artificial cells by modifying a defined protein expression system and demonstrated that artificial cells can show time development of spatiotemporal patterning similar to living cells. This system also allows generation and disappearance of spatiotemporal patterning, is controllable by a small molecule in artificial cells, and has the ability for application in cargo transporters. The system developed here provides a new material and a technique for understanding life, development of drug delivery tools, and creation of molecular robots.

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A dual system using compartmentalized partnered replication for selection of arsenic-responsive transcriptional regulator

Seaim Lwin Aye, Kei Fujiwara, Nobuhide Doi

The Journal of Biochemistry (Journal of Biochemistry) 164 ( 5 ) mvy055 - 348 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Engineering and design of genetic circuit in living cell is critical in accessing the beneficial application of synthetic biology. Directed evolution can avoid the complicated rational design of such circuit by screening or selecting functional circuit from non-functional one. Here, we proposed a positive–negative selection system for selecting a transcription factor that activates gene expression in response to arsenic in solution. First, we developed a whole cell biosensor for sensing arsenite in liquid using a regulator (ArsR) and a reporter (GFP), and evaluated its performance. Second, we developed a positive selection system for active ArsR using compartmentalized partnered replication that uses thermostable DNA polymerase as the reporter of activity. Third, we developed a negative selection system using sucrose-induced suicide gene SacB as the reporter for exclusion of inactive ArsR variants.

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Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Aye Seaim Lwin, Fujiwara Kei, Ueki Asuka, Doi Nobuhide

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 499 ( 2 ) 170 - 176 2018.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

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Increasing Elasticity through Changes in the Secondary Structure of Gelatin by Gelation in a Microsized Lipid Space

Sakai Atsushi, Murayama Yoshihiro, Fujiwara Kei, Fujisawa Takahiro, Sasaki Saori, Kidoaki Satoru, Yanagisawa Miho

ACS Central Science (ACS Central Science) 4 ( 4 ) 477 - 483 2018.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2374-7943

Even though microgels are used in a wide variety of applications, determining their mechanical properties has been elusive because of the difficulties in analysis. In this study, we investigated the surface elasticity of a spherical microgel of gelatin prepared inside a lipid droplet by using micropipet aspiration. We found that gelation inside a microdroplet covered with lipid membranes increased Young's modulus E toward a plateau value E∗ along with a decrease in gel size. In the case of 5.0 wt % gelatin gelled inside a microsized lipid space, the E∗ for small microgels with R ≤ 50 μm was 10-fold higher (35-39 kPa) than that for the bulk gel (∼3 kPa). Structural analysis using circular dichroism spectroscopy and a fluorescence indicator for ordered beta sheets demonstrated that the smaller microgels contained more beta sheets in the structure than the bulk gel. Our finding indicates that the confinement size of gelling polymers becomes a factor in the variation of elasticity of protein-based microgels via secondary structure changes.

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Single Micrometer-Sized Gels: Unique Mechanics and Characters for Applications.

Yanagisawa Miho, Watanabe Chiho, Fujiwara Kei

Gels (MDPI) 4 ( 2 ) 29 2018.03

Research paper (scientific journal), Joint Work, Accepted

Microgels—small gels of submicron to micron size—are widely used in food, cosmetics and biomedical applications because of their biocompatibility and/or fast response to external environments. However, the properties of “single” microgels have not been characterized due to limitations in preparation technologies and measurement methods for single microgels with sizes in the multi-micrometer range. The synthesis of multiple shapes of single microgels and their characterization are important for further functionalization and application of gel-based materials. In this review, we explain the recent advancements in microgel fabrication and characterization methods for single microgels. The first topic discussed includes the self-assembly methods for single microgel fabrication using physical phenomena such as phase separation, interfacial wetting and buckling instability. The second topic deals with methods for analyzing the mechanics of single microgels and the differences between their mechanical characteristics and those of bulk gels. The recent progress in the fabrication and characterization of single microgels will bring important insights to the design and functionalization of gel-based materials.

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Artificial Cell Fermentation as a Platform for Highly Efficient Cascade Conversion

Fujiwara Kei, Adachi Takuma, Doi Nobuhide

ACS Synthetic Biology (ACS Synthetic Biology) 7 ( 2 ) 363 - 370 2018.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 2161-5063

Because of its high specificity and stereoselectivity, cascade reactions using enzymes have been attracting attention as a platform for chemical synthesis. However, the sensitivity of enzymes outside their optimum conditions and their rapid decrease of activity upon dilution are drawbacks of the system. In this study, we developed a system for cascade enzymatic conversion in bacteria-shaped liposomes formed by hypertonic treatment, and demonstrated that the system can overcome the drawbacks of the enzymatic cascade reactions in bulk. This system produced final products at a level equivalent to the maximum concentration of the bulk system (0.10 M, e.g., 4.6 g/L), and worked even under conditions where enzymes normally lose their function. Under diluted conditions, the conversion rate of the artificial cell system was remarkably higher than that in the bulk system. Our results indicate that artificial cells can behave as a platform to perform fermentative production like microorganisms.

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Improvement of microtas for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Einaga Y., Matsumoto Y., Miki N., Fujiwara K., Doi N.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 (22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018) 4 2247 - 2249 2018

Here we report an improved μTAS based on electrochemical measurements and microbeads sorting for screening of NAD(P)-dependent oxidoreductases. By using this system, we measured the activity of model enzymes immobilized on microbeads by detection of NADH with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. Then, we recovered the magnetic microbeads or the droplets containing genes of enzymes with higher activity by magnetophoretic or dielectrophoretic sorting, respectively. Our μTAS should be useful for directed evolution of various NAD(P)-dependent oxidoreductases with high activity and stability in the future.

Universal glass-forming behavior of in vitro and living cytoplasm

Nishizawa Kenji, #HFujiwara Kei#IR, Ikenaga Masahiro, Nakajo Nobushige, Yanagisawa Miho, Mizuno Daisuke

Scientific Reports 7 ( 1 ) 15143 2017.12

Research paper (scientific journal), Joint Work, ISSN 2045-2322

Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of in vitro and living cytoplasm using the particle-tracking and manipulation technique. The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart.

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Liposomal internal viscosity affects the fate of membrane deformation induced by hypertonic treatment

Fujiwara Kei, Yanagisawa Miho

Soft Matter (Royal Society of Chemistry) 13 ( 48 ) 9192 - 9198 2017.11

Research paper (scientific journal), Joint Work, ISSN 1744-683X

Artificial lipid membranes have been utilized to understand the physical mechanisms of the deformation patterns of live cells. However, typical artificial membrane systems contain only dilute components compared to those in the cytoplasm of live cells. By using giant unilamellar liposomes containing dense protein solutions similar to those in live cells, we here reveal that viscosity derived from internal crowding affects the deformation patterns of lipid membranes. After hypertonic treatment, liposome deformation patterns transitioned from budding to tubing when the initial internal macromolecular concentrations were increased. Remarkably, instead of observing different transition concentrations between two species of macromolecules, the viscosity at the transition concentration was found to be similar. Further analyses clearly demonstrated that the internal viscosity affects the deformation patterns of lipid membranes induced by hypertonic treatment. These results indicate that the viscosity of the cytoplasm is a key factor in determining cell deformation, and suggest the association of a process involving dynamic instability, such as a viscous fingering phenomenon, during the determination of deformation patterns by hypertonic treatment.

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In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile

Fujiwara Kei, Sawamura Tsunehito, Niwa Tatsuya, Deyama Tatsuki, Nomura Shin Ichiro M., Taguchi Hideki, Doi Nobuhide

Nucleic Acids Research 45 ( 19 ) 11449 - 11458 2017.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.

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DNA cytoskeleton for stabilizing artificial cells

Kurokawa Chikako, Fujiwara Kei, Morita Masamune, Kawamata Ibuki, Kawagishi Yui, Sakai Atsushi, Murayama Yoshihiro, Nomura M. Shin-ichiro, Murata Satoshi, Takinoue Masahiro, and Yanagisawa　Miho

Proceedings of the National Academy of Sciences (National Academy of Sciences) 114 ( 28 ) 7228 - 7233 2017.06

Research paper (scientific journal), Joint Work, Accepted

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

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Human-derived fusogenic peptides for the intracellular delivery of proteins

Sudo Kei, Niikura Keisuke, Iwaki Kouta, Kohyama Shunshi, Fujiwara Kei, Doi Nobuhide

Journal of Controlled Release 255 1 - 11 2017.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0168-3659

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322–340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and β-galactosidase were delivered efficiently and retained their activities.

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In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display

Nakayama Masanao, Komiya Shoko, FUJIWARA Kei, Horisawa kenichi, Doi Nobuhide

Biochem. Biophys. Res. Comm. 478 ( 2 ) 606 - 611 2016.09

Research paper (scientific journal), Joint Work, Accepted

Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption

FUJIWARA Kei, Doi Nobuhide

PLOS ONE 11 ( 4 ) e0154614 2016.04

Research paper (scientific journal), Joint Work, Accepted

Identification of novel in vivo obligate GroEL/ES substrates based on data from a cell-free proteomics approach.

Niwa Tatsuya, FUJIWARA Kei, Taguchi hideki

FEBS Lett. 590 ( 2 ) 251 - 257 2016.01

Research paper (scientific journal), Joint Work, Accepted

PURE mRNA display for in vitro selection of single-chain antibodies.

Nagumo Yu, FUJIWARA Kei, Horisawa Kenichi, Yanagawa Hiroshi, Doi Nobuhide

J. Biochem. 159 ( 5 ) 519 - 526 2015.12

Research paper (scientific journal), Joint Work, Accepted

Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions

Morita Masamune, Onoe Hiroaki, Yanagisawa Miho, Ito Hiroaki, Ichikawa Masatoshi, Fujiwara Kei, Saito Hirohide, Takinoue Masahiro

ChemBioChem 16 ( 14 ) 2029 - 2035 2015.09

Research paper (scientific journal), Joint Work, ISSN 1439-4227

We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2, μL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems. Shoot to fill: We have developed a simple and robust microfluidic method for the generation of cell-sized liposomes with controlled lipid composition. Microdroplets are generated by centrifugal microdroplet shooting then filtering to form liposomes. We believe that this method can be applied to the study of cell-sized liposomes and the construction of artificial cell-like systems.

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Generation of monodisperse cell-sized microdroplets using a centrifuge-based axisymmetric co-flowing microfluidic device

Hitoyoshi Yamashita, Masamune Morita, Haruka Sugiura, Kei Fujiwara, Hiroaki Onoe, Masahiro Takinoue

Journal of Bioscience and Bioengineering (Elsevier) 119 ( 4 ) 492 - 495 2015.04

Research paper (scientific journal), Joint Work, Accepted

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Peptide sequences converting polyglutamine into a prion in yeast

Wataru Odani, Kazuhiro Urata, Momoko Okuda, Shunsuke Okuma, Hiroko Koyama, Chan‐Gi Pack, Kei Fujiwara, Tatsuya Nojima, Masataka Kinjo, Shigeko Kawai‐Noma, Hideki Taguchi

FEBS Journal 282 ( 3 ) 477 - 490 2015.02

Research paper (scientific journal), Joint Work, Accepted

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Development of μtas for high-throughput screening of nad(p)-dependent oxidoreductases

Shimokihara Shota, Shitara Shunya, Oyobiki Ryo, Watanabe Takeshi, Einaga Yasuaki, Matsumoto Yoshinori, Fujiwara Kei, Horisawa Kenichi, Doi Nobuhide

1302 - 1304 2015

Research paper (international conference proceedings), Joint Work

Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activity is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we developed a microfluidic total analysis system for high-throughput screening of NAD(P)-dependent oxidoreductases. This system comprises three microfluidic devices: an encapsulating device, a NADH measuring device, and a magnetic beads sorting device. We demonstrated the utility of each part of the system.

Conversion of a Chaperonin GroEL-Independent Protein into an Obligate Substrate

ISHIMOTO takuya, FUJIWARA Kei, NIWA Tatsuya, TAGUCHI hideki

The Journal of Biological Chemistry (The American Society for Biochemistry and Molecular Biology, Inc.) 289 32073 - 32080 2014.11

Research paper (scientific journal), Joint Work

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Multiple patterns of polymer gels in microspheres due to the interplay among phase separation, wetting, and gelation

Miho Yanagisawa, Shinpei Nigorikawa, Takahiro Sakaue, Kei Fujiwara, and Masayuki Tokita

Proceedings of the National Academy of Sciences (National Acad Sciences) 111 ( 45 ) 15894 - 15899 2014.11

Research paper (scientific journal), Joint Work, Accepted

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Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion

Akira C Saito, Toshihiko Ogura, Kei Fujiwara, Satoshi Murata, Shin-ichiro M Nomura

PloS one 9 e106853 2014.09

Research paper (scientific journal), Joint Work, Accepted

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Generation of Giant Unilamellar Liposomes Containing Biomacromolecules at Physiological Intracellular Concentrations using Hypertonic Conditions.

Yanagisawa, Miho, Fujiwara, Kei

ACS Synthetic Biology 3 ( 12 ) 870 - 874 2014.02

Research paper (scientific journal), Joint Work, Accepted

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DNAナノテクノロジーのためのアウトリーチツールの開発と評価

吉澤慧, 藤原慶, 野村 M.慎一郎, 村田智

形の科学会誌 (形の科学会) 29 ( 2 ) 2014

Research paper (scientific journal), Joint Work

The rapid synthesis of cell-sized liposomes by centrifuge-based microfluidic device

Morita Masamune, Onoe Hiroaki, Yanagisawa Miho, Fujiwara Kei, Saito Hirohide, Takinoue Masahiro.

18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 (Chemical and Biological Microsystems Society) 336 - 338 2014

Research paper (international conference proceedings), Joint Work

細胞内濃厚環境に近づいた無細胞発現系

FUJIWARA Kei, Nomura M. Shin-ichiro

生物物理 53 ( 5 ) 262 - 263 2013.09

Joint Work, Accepted

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Molecular robotics approach for constructing an artificial cell model

Shin-Ichiro M Nomura, Yusuke Sato, Kei Fujiwara

Advances in Artificial Life, ECAL 12 488 - 491 2013.09

Research paper (international conference proceedings), Joint Work, Accepted

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Construction of a remote-controlled supramolecular micro-crawler

Daiki Komatsu, Kei Fujiwara, Shin-Ichiro M Nomura

Advances in Artificial Life, ECAL 12 208 - 209 2013.09

Research paper (international conference proceedings), Joint Work, Accepted

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Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

Fujiwara, Kei, Katayama, Tsutomu, Nomura, Shin-ichiro M.

NUCLEIC ACIDS RESEARCH 41 ( 14 ) 7176 - 7183 2013.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

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Physicochemical Analysis from Real-Time Imaging of Liposome Tubulation Reveals the Characteristics of Individual F-BAR Domain Proteins

Tanaka-Takiguchi, Yohko, Itoh, Toshiki, Tsujita, Kazuya, Yamada, Shunsuke, Yanagisawa, Miho, Fujiwara, Kei, Yamamoto, Akihisa, Ichikawa, Masatoshi, Takiguchi, Kingo

LANGMUIR 29 ( 1 ) 328 - 336 2013.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0743-7463

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Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

Fujiwara, Kei, Nomura, Shin-ichiro M.

PLOS ONE 8 ( 1 ) 1552 - 1564 2013.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

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A bacterial salt sensor created by multiplying phenotypes of GroE-depleted Escherichia coli

Fujiwara, Kei, Aoi, Keita B., Nomura, Shin-ichiro M.

ANALYTICAL METHODS 5 ( 21 ) 5918 - 5922 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 1759-9660

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Mechanism of methionine synthase overexpression in chaperonin-depleted Escherichia coli

Fujiwara, Kei, Taguchi, Hideki

MICROBIOLOGY-SGM 158 917 - 924 2012.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1350-0872

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Cell-Sized confinement in microspheres accelerates the reaction of gene expression

Kato, Ayako, Yanagisawa, Miho, Sato, Yuko T., Fujiwara, Kei, Yoshikawa, Kenichi

SCIENTIFIC REPORTS 2 5918 - 5922 2012.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

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A systematic survey of in vivo obligate chaperonin-dependent substrates

Fujiwara, Kei, Ishihama, Yasushi, Nakahigashi, Kenji, Soga, Tomoyoshi, Taguchi, Hideki

EMBO JOURNAL 29 ( 9 ) 1552 - 1564 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0261-4189

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Filamentous morphology in GroE-depleted Escherichia coli induced by impaired folding of FtsE

Fujiwara, Kei, Taguchi, Hideki

JOURNAL OF BACTERIOLOGY 189 ( 16 ) 5860 - 5866 2007.08

Research paper (scientific journal), Accepted, ISSN 0021-9193

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Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus

Fujiwara, Kei, Tsubouchi, Taishi, Kuzuyama, Tomohisa, Nishiyama, Makoto

MICROBIOLOGY-SGM 152 3585 - 3594 2006.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 1350-0872

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